The present invention relates to novel phenylalanine ammonia-lyase producing microorganisms and methods for their selection, production, and use. More particularly, the invention concerns microorganisms which produce relatively high levels of the enzyme, phenylalanine ammonia lyase (hereinafter sometimes called PAL), which in turn, is useful for the production of L-phenylalanine.
L-phenylalanine is an essential amino acid in man, and is, therefore, an important ingredient of enteral and parenteral nutritional formulations. In addition, this amino acid is useful as a starting material for the production of other products, such as the artificial sweetener, aspartame. Various microbial processes for the production of phenylalanine are known. For example, U.S. Pat. No. 3,660,235 describes the production of phenylalanine by phenylalanine analogue resistant strains of Brevibacterium, Corynebacterium, Arthrobacter, Bacillus and Candida. The production of this amino acid by tyrosine-requiring mutants of certain strains of Brevibacterium, Corynebacterium, Arthrobacter,and Escherichia are also known. See U.S. Pat. No. 3,654,079 and 3,909,353.
PAL catalyzes the breakdown of L-phenylalanine to trans-cinnamic acid and ammonia. This enzymatic reaction is reversible, and British Pat. No. 1,489,468 discloses a process for the production of L-phenylalanine which involves the PAL-catalyzed reaction of trans-cinnamic acid with ammonium ions to yield L-phenylalanine. This reaction has been found to be a useful procedure for producing L-phenylalanine, and therefore, there is a continuing need to obtain production microorganisms which produce high levels of PAL activity. Such microorganisms can be used directly for the conversion of cinnamic acid and ammonium ions to L-phenylalanine, or the enzyme can be isolated from the cells and used to produce L-phenylalanine in various forms of bioreactors.
The present invention concerns novel microorganisms which overproduce PAL. These microorganisms are obtained by conventional mutation techniques. Since PAL-containing cells can derive energy from the enzymatic conversion of phenylalanine to trans-cinnamic acid and ammonia and the subsequent metabolism of trans-cinnamic acid, a means for selecting PAL-overproducing mutants involves growing such cells on minimal nutritional media containing L-phenylalanine as substantially the sole carbon source. Although this selection technique can be used for identifying PAL-producing mutants, it has been found that the degradation of L-phenylalanine occurs much more rapidly than subsequent degradative reactions, therefore, the technique is not particularly useful for differentiating those mutants which produce high levels of PAL activity from only nominally productive strains.